ABSTRACT There is a critical need to discover and develop safe and effective antibiotics for the treatment of multidrug- resistant bacterial infections. Each year approximately 1.5 million patients in the US get hospital acquired infections (HAIs), with a mortality rate of ~6%. Nearly 70% of the bacteria causing HAIs are resistant to at least one commonly used antibiotic. Natural products (NPs) and their derivatives represent ~50% of FDA approved small molecule drugs over the past 5 decades including many important antibiotics. The traditional NP activity- guided fractionation approach is labor intensive and time-consuming, and often leads to rediscovery of known compounds or unwanted nuisance compounds. Genome-mining, using state of the art bioinformatic sequence analysis, can identify and evaluate the novelty of NP gene clusters early in the discovery process and is quickly replacing the traditional approach. Warp Drive Bio has sequenced the genomes of over 135,000 actinomycete strains from diverse sources worldwide to generate a proprietary genomic database that contains approximately ~3.5 million NP biosynthetic gene clusters. Importantly ~75% of cluster families identified in our database have yet to be reported in the literature. We have identified ~200 biosynthetic ?-lactams (BLs) gene clusters predicted to encode for synthesis of novel BL structures, thus providing an unprecedented opportunity to discover BLs possessing new antimicrobial activities. We have also developed synthetic biology tools to turn on the biosynthetic gene clusters which otherwise might be expressed at levels below the detection limit or not expressed at all. In addition, we have initiated development of a novel, BL specific, and sensitive mass spectrometer (MS) based ligand-binding assay for discovery of novel BLs. During our initial experiments, we observed a complex formed between a penicillin binding protein (Pseudomonas aeruginosa PBP3) and various BL ligands, and between a ?-lactamase (E. coli TEM1) and carbapenems. However, in some experiments the assay sensitivity was decreased significantly due to impurities in the crude extracts and catalytic activity of ?-lactamases. In this Phase I application, we aim to improve assay sensitivity using various chromatographic separation methods prior the LC-MS analysis, and by utilization of mutant ?-lactamases defective in deacylation activity. Mutant TEM1(E166N), CTX-M15 (E166N) and KPC2 (E166N) can covalently interact with BLs at the catalytic serine residue but will not efficiently hydrolyze the ring opened BLs, allowing the acyl-protein adduct to be captured yielding increased LC-MS detection sensitivity. This improved LC-MS ligand binding assay (using PBPs and ?- lactamase) should enable the rapid mass-based detection and identification of new BLs in crude extracts, thus increasing the likelihood of success for discovery of novel compounds/bio-activities encoded by specific BL gene clusters.